Cholesterol sulfate alleviates ulcerative colitis by promoting cholesterol biosynthesis in colonic epithelial cells

Cholesterol sulfate, produced by hydroxysteroid sulfotransferase 2B1 (SULT2B1), is highly abundant in the intestine. Herein, we study the functional role and underlying intestinal epithelial repair mechanisms of cholesterol sulfate in ulcerative colitis. The levels of cholesterol and cholesterol sulfate, as well as the expression of Sult2b1 and genes involved in cholesterol biosynthesis, are significantly higher in inflamed tissues from patients with ulcerative colitis than in intestinal mucosa from healthy controls. Cholesterol sulfate in the gut and circulation is mainly catalyzed by intestinal epithelial SULT2B1. Specific deletion of the Sult2b1 gene in the intestinal epithelial cells aggravates dextran sulfate sodium-induced colitis; however, dietary supplementation with cholesterol sulfate ameliorates this effect in acute and chronic ulcerative colitis in mice. Cholesterol sulfate promotes cholesterol biosynthesis by binding to Niemann-Pick type C2 protein and activating sterol regulatory element binding protein 2 in colonic epithelial cells, thereby alleviates ulcerative colitis. In conclusion, cholesterol sulfate contributes to the healing of the mucosal barrier and exhibits therapeutic efficacy against ulcerative colitis in mice.

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The transcriptional expressions of mouse Npc1l1 and human SULT2B1, HMGCS1, MVK, MVD, FDPS, FDFT1, SQLE, LSS, CYP51A1, MSMO1, NSDHL, SC5D, and DHCR7 were analyzed from the published GEO datasets (http://www.ncbi.nlm.nih.gov/geo/). The gene expression profiles of the mouse jejunum, ileum, and colon were obtained from GEO (GSE 143342). The RNA-seq data from patients with clinical UC were from GEO (GSE 111889). Raw data of RNA sequencing generated in this study were deposited at SRA database of NCBI with the accession number PRJNA644070. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database used in the study are available at http://geneontology.org/ and https://www.genome.jp/kegg/. Gene set enrichment analysis (GSEA) was performed using the GSEA software (http://software.broadinstitute.org/gsea/index.jsp). If needed, contact X.L. for original data described in the paper. Contact X.L.
for requesting Sult2b1f/f and Sult2b1#IEC mouse strain, and all other plasmids or reagents described in this article. Source data are provided with this paper.
Sample size estimates has been performed on previous experience to obtain statistical significance and reproducibility. For in vivo studies, the sample size was determined to be enough to obtain the statistical difference between groups, and genotype. Animals were randomly assigned to treatment groups. All sample sizes are listed in the corresponding figure legends or on the figures. All experiments were repeated at least three times.
When we compared the normalized FPKM of genes of interest in the rectum among non-IBD (n=23) and UC groups(n=26) from published RNA-seq data (GSE 111889), we found the normalized FPKM of SULT2B1 in non-IBD sample "PSM6XBZW" is 0.02416, which is 19.02-fold of the average value of other 22 non-IBD samples (average: 0.00127, SD: 0.00054). Then, when we compared gene expressions in figure 1a and figure 3f, we excluded this outlier (PSM6XBZW) from the non-IBD group in this study. Therefore, 22 non-IBD and 26 UC samples were included for analysis in this study.
All experiments were conducted at least three times independently, and similar results were adopted for further analysis to guarantee reproducibility.
Samples were randomly allocated into the study.
This study included a lot of complicated experimental design, the feasibility of blinding was poor, thus blinding was not efficiently applied. The investigators were blinded to group allocation during data collection. Data analysis were performed by different investigators and analysis to avoid conscious and unconscious bias.

March 2021
Materials & experimental systems Validation of the use of anti-human SULT2B1 antibody for human in Western blot and immunohistochemistry has been provided by the manufacturer's website.
Validation of the use of anti-mouse SULT2B1 antibody for mouse in Western blot has been provided by the manufacturer's website.
Validation of the use of SREBP2 antibody for human in immunostaining has been provided by the manufacturer's website.
Validation of the use of HA antibody for human in Western blot has been provided by the manufacturer's website.
Validation of the use of SLC10A6 antibody for human in immunostaining have been provided by the manufacturer's website.
Validation of the use of "-actin and HSP90 for human and mouse in Western blot has been provided by the manufacturer's website.
Validation of the use of NPC2 for human in Western blot has been provided by the manufacturer's website.
Validation of the use of FLAG antibody for human in Western blot has been provided by the manufacturer's website.
Validation of the use of goat anti-rabbit secondary antibody, goat anti-mouse secondary antibody and peroxidase-conjugated rabbit anti-sheep secondary antibody in Western-blot has been provided by the manufacturers' website.
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All the cell lines were test negative for the mycoplasma contamination.
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The study did not involve field-collected samples  Supplementary Table 7.
Patients in this study were recruited from Shanxi Provincial People's Hospital, Taiyuan, China. All patients information and samples were collected based on clinical requirement for diagnosis. There were no self-selection bias or other biases.
All patients gave informed consents for collection of tissue collection. All procedures were performed in accordance with institutional guidelines and were approved by Shanxi Provincial People's Hospital Research Ethics Committee with the reference number 2019-70 for this study. The study was conducted in accordance with the criteria set by the Declaration of Helsinki.